The present invention relates to separating charged molecules, such as DNA, proteins, polypeptides, and other molecules or charged particles using a photoelectrochemical method.
Analysis of a sample of biological origin frequently requires the separation of mixtures, and biomolecules, such as nucleic acids, proteins, and polypeptides, which often have limited sample size. Electrophoresis, in which charged molecules move in a liquid under the influence of an electric field, has long been the method of choice for separating many classes of biomolecules. This method takes advantage of differing migration velocities, vep, of different molecules.
Migration velocity, vep, the distance the molecule or particle moves (L) per unit time (t), is the product of the electrophoretic mobility, μep, times the electric field strength E (units of volts/cm).vep=μep×E μep=q/6πηRwhere q is the charge on the particle and η is the viscosity of the medium. The velocity is, thus, directly proportional to the charge on the particle and the field strength and inversely proportional to the size of the particle and the viscosity of the medium. For relatively large particles or biomolecules, the charge increases as the size of the molecule increases, and the charge to mass (or radius) ratio becomes nearly constant. Under these circumstances if the electrophoresis is carried out in the presence of a gel composed of agrose or crosslinked polyacrylamide, the gel structure creates a molecular sieving effect that allows the molecules or particles to be separated on the basis of size.
Capillary gel electrophoresis is typically carried out in 50 μm diameter capillaries that are 10 cm to 1 m long with a field strength that is generally in the range of 100 V/cm to 500 V/cm, and requires a high-applied voltage greater than 1 KV. Heat generation is directly proportional to the square of the applied voltage, and the voltages required to achieve separation in capillary electrophoresis may cause degradation of sensitive samples.
Transport of biomolecules has been demonstrated when applying voltages less than 1 KV. Semiconductors such as Si, Ge, GaAs, TiO2, CdS, and ZnO in contact with a liquid exhibit a change in surface charge upon irradiation with light of an appropriate wavelength when electronic bands of the semiconductors are bent. These reactions occur initially by the absorption of photons of energies greater than the corresponding semiconductor band gap to form conduction band electron-valence band hole pairs. This phenomenon has been shown to be advantageous for the photoelectrophoretic transport of biomolecules, but prior to the present disclosure has not been used for separating biomolecules.
Band bending can be achieved by suitably polarizing the semiconductor with respect to the liquid with a power supply. The back contact to the semiconductor electrode is Ohmic in character while the semiconductor-liquid interface acts as a Schottky barrier. Therefore, most of the applied voltage is dropped at the semiconductor-liquid interface creating a space charge (depletion or accumulation) layer in the semiconductor. The formation of depletion or accumulation layer depends on the bias and the type of semiconductor. The nature of band bending can be changed from depletion to accumulation by changing the sign of the applied potential with respect to flat band potential of the semiconductor-liquid interface.
Irradiation of the semiconductor-liquid interface with photons of appropriate energy produces electron-hole pairs in the depletion or accumulation layer. The field in the depletion or accumulation layer separates the electron-hole pairs. For example, for a n-type semiconductor, the bands are bent down for a depletion layer, and therefore the electrons come to the semiconductor-liquid interface during illumination. In the case of an accumulation layer irradiation causes the holes to accumulate at the solid-liquid interface. The presence of a charge on the semiconductor can be used for attracting charged biomolecules.
An experiment conducted by Gurtner et al demonstrated that DNA oligonucleotides could be photoeletrophoretically transported to a stabilized semiconductor surface coated with a streptavidin-agarose permeation layer (C. Gurtner, C. Edman, R. Formosa, M. Heller, Photoelectrophoretic Transport and Hybridization of DNA Oligonucleotides on Unpatterned Silicon Substrates, Journal of the American Chemical Society, vol. 122, no. 36, (2000) pp 8589–8594.). In this experiment, micro-illumination of the surface generated photoelectrochemical currents that were used to electrophoretically transport biointylated DNA capture strands to arbitrarily selected locations for attachment. These experiments demonstrate that a photogenerated potential is sufficient to cause movement in biointylated DNA capture strands.
A subject of a considerable amount of research in recent years has been microscale fluid handling systems that perform fast, automated, high-resolution sample preparation, reaction, and separation. Currently, this is being accomplished through advances in microfluidics. The idea is that once the manipulation of fluids can be mastered on the microscale, key experiments for biomolecule separation and analysis can be integrated and automated—all on a mass-produced chip. In microfluidic-based devices, DNA, proteins, and other molecules are transported, manipulated, and separated through miniature channels embedded into the chip. Detection systems can also be integrated into the chip or affixed externally as a separate component for seamless, automated and highly sensitive detection.